Solid phase peptides synthesis processes and associated systems

ABSTRACT

Systems and processes for performing solid phase peptide synthesis are generally described. Solid phase peptide synthesis is a known process in which amino acid residues are added to peptides that have been immobilized on a solid support. In certain embodiments, the inventive systems and methods can be used to perform solid phase peptide synthesis quickly while maintaining high yields. Certain embodiments relate to processes and systems that may be used to heat, transport, and/or mix reagents in ways that reduce the amount of time required to perform solid phase peptide synthesis.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/833,745, filed Mar. 15, 2013, and entitled “Solid Phase PeptideSynthesis Processes and Associated Systems,” which is incorporatedherein by reference in its entirety for all purposes.

TECHNICAL FIELD

Systems and processes for performing solid phase peptide synthesis aregenerally described.

BACKGROUND

Solid phase peptide synthesis is a process used to chemically synthesizepeptides on solid supports. In solid phase peptide synthesis, an aminoacid or peptide is bound, usually via the C-terminus, to a solidsupport. New amino acids are added to the bound amino acid or peptidevia coupling reactions. Due to the possibility of unintended reactions,protection groups are typically used. To date, solid phase peptidesynthesis has become standard practice for chemical peptide synthesis.The broad utility of solid phase peptide synthesis has been demonstratedby the commercial success of automated solid phase peptide synthesizers.Though solid phase peptide synthesis has been used for over 30 years,fast synthesis techniques have not yet been developed. Accordingly,improved processes and systems are needed.

SUMMARY

Solid phase peptide synthesis processes and associated systems aregenerally described. Certain embodiments relate to systems and methodswhich can be used to perform solid phase peptide synthesis quickly whilemaintaining high yield. In some embodiments, reagents can be heated,transported, and/or mixed in ways that reduce the amount of timerequired to perform solid phase peptide synthesis. The subject matter ofthe present invention involves, in some cases, interrelated products,alternative solutions to a particular problem, and/or a plurality ofdifferent uses of one or more systems and/or articles.

In some embodiments, a process for adding amino acid residues topeptides is provided. The process comprises, in certain embodiments,providing a plurality of peptides comprising protection groups, eachpeptide immobilized on a solid support; exposing a deprotection reagentto the immobilized peptides to remove the protection groups from atleast a portion of the immobilized peptides; removing at least a portionof the deprotection reagent; exposing activated amino acids to theimmobilized peptides such that at least a portion of the activated aminoacids are bonded to the immobilized peptides to form newly-bonded aminoacid residues; and removing at least a portion of activated amino acidsthat do not bond to the immobilized peptides. In some embodiments, anamino acid residue is added to at least about 99% of the immobilizedpeptides during the amino acids exposing step. In certain embodiments,the total amount of time taken to perform the combination of all of thedeprotection reagent exposing step, the deprotection reagent removalstep, the activated amino acid exposing step, and the activated aminoacid removal step is about 10 minutes or less and the protection groupscomprise fluorenylmethyloxycarbonyl protection groups and/or the totalamount of time taken to perform the combination of all of thedeprotection reagent exposing step, the deprotection reagent removalstep, the activated amino acid exposing step, and the activated aminoacid removal step is about 5 minutes or less.

In certain embodiments, the process comprises flowing a first streamcomprising amino acids; flowing a second stream comprising an amino acidactivating agent; merging the first and second streams to form a mixedfluid comprising activated amino acids; and within about 30 secondsafter merging the first and second streams to form the mixed fluid,exposing the mixed fluid to a plurality of peptides immobilized on asolid support.

In some embodiments, the process comprises heating a stream comprisingamino acids such that the temperature of the amino acids is increased byat least about 1° C.; and exposing the heated amino acids to a pluralityof peptides immobilized on a solid support, wherein the heating step isperformed prior to and within about 30 seconds of exposing the heatedamino acids to the peptides.

In certain embodiments, the process comprises providing a plurality ofpeptides comprising protection groups, each peptide immobilized on asolid support; performing a first amino acid addition cycle comprisingexposing amino acids to the immobilized peptides such that an amino acidresidue is added to at least about 99% of the immobilized peptides; andperforming a second amino acid addition cycle comprising exposing aminoacids to the immobilized peptides such that an amino acid residue isadded to at least about 99% of the immobilized peptides. In someembodiments, the total amount of time between the ends of the first andsecond amino acid addition cycles is about 10 minutes or less and theprotection groups comprise fluorenylmethyloxycarbonyl protection groupsand/or the total amount of time between the ends of the first and secondamino acid addition cycles is about 5 minutes or less.

In certain embodiments, the process comprises providing a plurality ofpeptides immobilized on a solid support; and exposing activated aminoacids to the immobilized peptides such that at least a portion of theactivated amino acids are bonded to the immobilized peptides to formnewly-bonded amino acid residues; wherein an amino acid residue is addedto at least about 99% of the immobilized peptides within about 1 minuteor less.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 is a schematic illustration of a system for performing peptidesynthesis, according to one set of embodiments;

FIG. 2A is, according to certain embodiments, an exemplary schematicdiagram of a peptide synthesis system;

FIG. 2B is, according to certain embodiments, a photograph of anexemplary peptide synthesis system;

FIG. 2C is, according to certain embodiments, a chromatogram for asynthesized peptide Fmoc-ALFALFA-CONH₂;

FIG. 2D is, according to certain embodiments, an exemplary schematicdiagram of a reactor;

FIG. 3A is, according to one set of embodiments, chromatograms ofLYRAG-CONH₂ peptides synthesized with different activated amino acidexposing times;

FIG. 3B is, according to one set of embodiments, chromatograms ofFmoc-ALF-CONH₂ peptides synthesized with different activated amino acidexposing times;

FIG. 3C is, according to one set of embodiments, an exemplary synthetictimeline;

FIG. 4A is, according to certain embodiments, a chromatogram and massspectrum for ACP (65-74) peptides synthesized using HATU;

FIG. 4B is, according to certain embodiments, a chromatogram and massspectrum for ACP (65-74) peptides synthesized using HBTU;

FIG. 4C is, according to certain embodiments, a chromatogram and massspectrum for ACP (65-74) peptides synthesized under batch conditions;

FIG. 4D is, according to certain embodiments, a chromatogram and massspectrum for ACP (65-74) peptides synthesized under flow conditions;

FIG. 5A is, according to one set of embodiments, a chromatogram and massspectrum for PnIA (A10L) peptides;

FIG. 5B is, according to one set of embodiments, a chromatogram and massspectrum for synthesized HIV-1 PR (81-99) peptides;

FIG. 6A is, according to certain embodiments, a total ion currentchromatograph for GCF peptide synthesized under conditions 5, as shownin Table 1;

FIG. 6B is, according to certain embodiments, a total ion currentchromatograph for GCF peptide synthesized under conditions 7, as shownin Table 1;

FIG. 6C is, according to certain embodiments, a total ion currentchromatograph for GCF peptide synthesized under conditions 8, as shownin Table 1;

FIG. 6D is, according to certain embodiments, a total ion currentchromatograph for GCF peptide synthesized under conditions 4, as shownTable 1;

FIG. 6E is, according to certain embodiments, an exemplary total ioncurrent chromatograph for an authentic Gly-D-Cys-L-Phe sample;

FIG. 7A is, according to certain embodiments, an exemplary scheme forthe chemical ligation of an affibody protein from three peptidefragments;

FIG. 7B is, according to certain embodiments, a chromatogram and massspectrum for a first affibody fragment;

FIG. 7C is, according to certain embodiments, a chromatogram and massspectrum for a second affibody fragment;

FIG. 7D is, according to certain embodiments, a chromatogram and massspectrum for a third affibody peptide fragment;

FIG. 7E is, according to certain embodiments, a chromatogram and massspectrum for the purified affibody;

FIG. 8A is, according to certain embodiments, a total ion chromatogramfor the N-terminal affibody fragment for a flow arrangement using BocN-terminal protecting groups;

FIG. 8B is, according to certain embodiments, a total ion chromatogramfor the N-terminal affibody fragment for a manual arrangement using BocN-terminal protecting groups;

FIG. 8C is, according to certain embodiments, a total ion chromatogramfor the middle affibody fragment for a flow arrangement;

FIG. 8D is, according to certain embodiments, a total ion chromatogramfor the middle affibody fragment for a manual arrangement;

FIG. 8E is, according to certain embodiments, a total ion chromatogramfor the C-terminal affibody fragment for affibody peptides for a flowarrangement;

FIG. 8F is, according to certain embodiments, a total ion chromatogramfor the C-terminal affibody fragment for affibody peptides for a manualarrangement;

FIG. 9A is, according to certain embodiments, a total ion chromatogramfor a purified first affibody fragment;

FIG. 9B is, according to certain embodiments, a total ion chromatogramfor a purified second affibody fragment;

FIG. 9C is, according to certain embodiments, a total ion chromatogramfor a purified third affibody fragment;

FIG. 9D is, according to certain embodiments, a total ion chromatogramfor the purified affibody fragment from the ligation of the first andsecond fragment;

FIG. 9E is, according to certain embodiments, a chromatogram and massspectrum for the purified affibody;

FIG. 10 is a plot of ultraviolet absorbance as a function of timerecorded during the synthesis of a peptide, according to one set ofembodiments;

FIG. 11 is a graph of flow rate versus wash time, according to one setof embodiments;

FIG. 12A is, according to one set of embodiments, a photograph of aninlet (left) and outlet (right);

FIG. 12B is, according to one set of embodiments, a photograph of aspacer;

FIG. 12C is, according to one set of embodiments, a photograph of areactor body unit;

FIG. 12D is, according to one set of embodiments, a photograph of anassembled reactor;

FIG. 12E is, according to one set of embodiments, a schematic of thereactor showing the reactor body, frit, and spacer;

FIG. 13 is a schematic illustration of an exemplary system forperforming peptide synthesis, according to one set of embodiments;

FIG. 14 is, according to certain embodiments, an exemplary total ionchromatogram of a synthesized peptide ALFALFA-CONHNH₂; and

FIG. 15 shows two exemplary chromatograms of peptides made using certainof the protein synthesis systems described herein.

DETAILED DESCRIPTION

Systems and processes for performing solid phase peptide synthesis aregenerally described. Solid phase peptide synthesis is a known process inwhich amino acid residues are added to peptides that have beenimmobilized on a solid support. In certain embodiments, the inventivesystems and methods can be used to perform solid phase peptide synthesisquickly while maintaining high yields. Certain embodiments relate toprocesses and systems that may be used to heat, transport, and/or mixreagents in ways that reduce the amount of time required to performsolid phase peptide synthesis.

Certain embodiments relate to a process for adding amino acid(s) to animmobilized peptide. FIG. 1 is a schematic illustration of an exemplarysystem 5 which can be used to perform certain of the inventive processesdescribed herein. The systems and methods described herein (system 5 inFIG. 1) can, in certain embodiments, involve flow-based synthesis (asopposed to batch-based synthesis, which is employed in many traditionalsolid phase peptide synthesis systems). In some such embodiments,continuous peptide synthesis can be performed, in which fluid (of oneform or another) is substantially continuously transported over theimmobilized peptides. For example, reagents and rinsing fluids may bealternatively and continuously transported over the immobilizedpeptides, in certain embodiments.

In some embodiments, peptides 20 may be immobilized on a solid support15. Solid support 15 may be contained within a vessel, such as reactor10. In some embodiments, and as shown in FIG. 1, a plurality of reagentreservoirs may be located upstream of and fluidically connected toreactor 10. In some embodiments, a reagent reservoir 25 contains aminoacids (e.g., pre-activated amino acids and/or amino acids that are notfully activated). In some instances, a reagent reservoir 26 contains anamino acid activating agent (e.g., an alkaline liquid, a carbodiimide,and/or a uronium activating agent), capable of completing the activationof the amino acids. In certain embodiments, a reagent reservoir 27contains a deprotection reagent, such as piperidine or trifluoroaceticacid. A reagent reservoir 28 may contain a solvent, such asdimethylformamide (DMF), that may be used, e.g., in a reagent removalstep. While single reservoirs have been illustrated in FIG. 1 forsimplicity, it should be understood that in FIG. 1, where singlereservoirs are illustrated, multiple reservoirs (e.g., each containingdifferent types of amino acids, different types of deprotection agents,etc.) could be used in place of the single reservoir.

In certain embodiments, peptides 20 comprise protection groups, forexample, on the N-termini of the peptides. As used herein, the term“protection group” is given its ordinary meaning in the art. Protectiongroups include chemical moieties that are attached to or are configuredto be attached to reactive groups (i.e., the protected groups) within amolecule (e.g., peptides) such that the protection groups prevent orotherwise inhibit the protected groups from reacting. Protection mayoccur by attaching the protection group to the molecule. Deprotectionmay occur when the protection group is removed from the molecule, forexample, by a chemical transformation which removes the protectiongroup.

In some embodiments, a plurality of peptides comprising protectiongroups may be bound to a solid support such that each peptide isimmobilized on the solid support. For example, the peptides may be boundto the solid support via their C termini, thereby immobilizing thepeptides.

In some embodiments, the process of adding amino acid residues toimmobilized peptides comprises exposing a deprotection reagent to theimmobilized peptides to remove at least a portion of the protectiongroups from at least a portion of the immobilized peptides. Thedeprotection reagent exposure step can be configured, in certainembodiments, such that side-chain protection groups are preserved, whileN-termini protection groups are removed. For instance, in certainembodiments, the protection group used to protect the peptides comprisesfluorenylmethyloxycarbonyl (Fmoc). In some such embodiments, adeprotection reagent comprising piperidine (e.g., a piperidine solution)may be exposed to the immobilized peptides such that the Fmoc protectiongroups are removed from at least a portion of the immobilized peptides.In some embodiments, the protection group used to protect the peptidescomprises tert-butyloxycarbonyl (Boc). In some such embodiments, adeprotection reagent comprising trifluoroacetic acid may be exposed tothe immobilized peptides such that the Boc protection groups are removedfrom at least a portion of the immobilized peptides. In some instances,the protection groups (e.g., tert-butoxycarbonyl, i.e., Boc) may bebound to the N-termini of the peptides.

In some embodiments, the process of adding amino acid residues toimmobilized peptides comprises removing at least a portion of thedeprotection reagent. In some embodiments, at least a portion of anyreaction byproducts (e.g., protection groups) that may have formedduring the deprotection step can be removed. In some instances, thedeprotection reagent (and, in certain embodiments byproducts) may beremoved by washing the peptides, solid support, and/or surrounding areaswith a fluid (e.g., a liquid such as an aqueous or non-aqueous solvent,a supercritical fluid, and the like), for example stored in optionalreservoir 28. In some instances, removing the deprotection reagent andreaction byproducts may improve the performance of subsequent steps(e.g., by preventing side reactions).

The process of adding amino acid residues to immobilized peptidescomprises, in certain embodiments, exposing activated amino acids to theimmobilized peptides such that at least a portion of the activated aminoacids are bonded to the immobilized peptides to form newly-bonded aminoacid residues. For example, the peptides may be exposed to activatedamino acids that react with the deprotected N-termini of the peptides.In certain embodiments, amino acids can be activated for reaction withthe deprotected peptides by mixing an amino acid-containing stream withan activation agent stream, as discussed in more detail below. In someinstances, the amine group of the activated amino acid may be protected,such that addition of the amino acid results in an immobilized peptidewith a protected N-terminus.

In some embodiments, the process of adding amino acid residues toimmobilized peptides comprises removing at least a portion of theactivated amino acids that do not bond to the immobilized peptides. Insome embodiments, at least a portion of the reaction byproducts that mayform during the activated amino acid exposure step may be removed. Insome instances, the activated amino acids and byproducts may be removedby washing the peptides, solid support, and surrounding areas with asolvent.

It should be understood that the above-referenced steps are exemplaryand an amino acid addition cycle need not necessarily comprise all ofthe above-referenced steps. Generally, an amino acid addition cycleincludes any series of steps that results in the addition of an aminoacid residue to a peptide.

In certain embodiments, during the amino acid addition steps, adding theamino acid can result in the peptide incorporating a single additionalamino acid residue (i.e., a single amino acid residue can be added tothe immobilized peptides such that a given peptide includes a singleadditional amino acid residue after the addition step). In some suchembodiments, subsequent amino acid addition steps can be used to buildpeptides by adding amino acid residues individually until the desiredpeptide has been synthesized. In some embodiments, more than one aminoacid residue (e.g., in the form of a peptide) may be added to a peptideimmobilized on a solid support (i.e., a peptide comprising multipleamino acid residues can be added to a given immobilized peptide).Addition of peptides to immobilized peptides can be achieved throughprocesses know to those of ordinary skill in the art (e.g., fragmentcondensation, chemical ligation). That is to say, during the amino acidaddition step, adding an amino acid to an immobilized peptide cancomprise adding a single amino acid residue to an immobilized peptide oradding a plurality of amino acid residues (e.g., as a peptide) to animmobilized peptide.

In some embodiments, amino acids can be added to peptides significantlyfaster than conventional methods. In certain embodiments, the totalamount of time taken to perform the combination of steps may beinfluenced by the protection group. For instance, in certain embodimentsin which the protection groups comprise fluorenylmethyloxycarbonyl(Fmoc), the total amount of time taken to perform the combination of allof the deprotection reagent exposing step, the deprotection reagentremoval step, the activated amino acid exposing step, and the activatedamino acid removal step is about 10 minutes or less, about 9 minutes orless, about 8 minutes or less, about 7 minutes or less, about 6 minutesor less, about 5 minutes or less, about 4 minutes or less, about 3minutes or less, about 2 minutes or less, about 1 minute or less, fromabout 10 seconds to about 10 minutes, from about 10 seconds to about 9minutes, from about 10 seconds to about 8 minutes, from about 10 secondsto about 7 minutes, from about 10 seconds to about 6 minutes, from about10 seconds to about 5 minutes, from about 10 seconds to about 4 minutes,from about 10 seconds to about 3 minutes, from about 10 seconds to about2 minutes, or from about 10 seconds to about 1 minute. In certainembodiments (including embodiments in which the protection groupscomprise tert-butyloxycarbonyl (Boc), fluorenylmethyloxycarbonyl (Fmoc),and/or other types of protection groups), the total amount of time takento perform the combination of all of the deprotection reagent exposingstep, the deprotection reagent removal step, the activated amino acidexposing step, and the activated amino acid removal step is about 5minutes or less, about 4 minutes or less, about 3 minutes or less, about2 minutes or less, about 1 minute or less, from about 10 seconds toabout 5 minutes, from about 10 seconds to about 4 minutes, from about 10seconds to about 3 minutes, from about 10 seconds to about 2 minutes, orfrom about 10 seconds to about 1 minute.

In general, the total amount of time taken to perform the combination ofall of the deprotection reagent exposing step, the deprotection reagentremoval step, the activated amino acid exposing step, and the activatedamino acid removal step is calculated by adding the amount of time ittakes to perform the deprotection reagent exposing step to the amount oftime it take to perform the deprotection reagent removal step and to theamount of time it take to perform the activated amino acid exposing stepand to the amount of time it takes to perform the activated amino acidremoval step.

In certain embodiments, the first amino acid addition step (and/orsubsequent amino acid addition steps) may add amino acids at arelatively high yield. For example, certain embodiments include exposingamino acids to the immobilized peptides such that an amino acid residueis added to at least about 99%, at least about 99.9%, at least about99.99%, or substantially 100% of the immobilized peptides. In certainembodiments, a second (and, in some embodiments, a third, a fourth, afifth, and/or a subsequent) amino acid addition cycle can be performedwhich may include exposing amino acids to the immobilized peptides suchthat an amino acid residue is added to at least about 99%, at leastabout 99.9%, at least about 99.99%, or substantially 100% of theimmobilized peptides. In certain embodiments, the use of processes andsystems of the present invention may allow the addition of more than oneamino acid to the immobilized peptides to occur relatively quickly(including within any of the time ranges disclosed above or elsewhereherein), while maintaining a high reaction yield.

In certain embodiments, one or more amino acid addition steps can beperformed while little or no double incorporation (i.e., adding multiplecopies of a desired amino acid (e.g., single amino acid residues orpeptides) during a single addition step). For example, in certainembodiments, multiple copies of the desired amino acid are bonded tofewer than about 1% (or fewer than about 0.1%, fewer than about 0.01%,fewer than about 0.001%, fewer than about 0.0001%, fewer than about0.00001%, or substantially none) of the immobilized peptides during afirst (and/or second, third, fourth, fifth, and/or subsequent) aminoacid addition step.

In some embodiments, multiple amino acid addition cycles can beperformed. Performing multiple amino acid addition cycles can result inmore than one single-amino-acid residue (or more than one peptide,and/or at least one single-amino-acid residue and at least one peptide)being added to a peptide. In certain embodiments a process for addingmore than one amino acid to immobilized peptides may comprise performinga first amino acid addition cycle to add a first amino acid andperforming a second amino acid addition cycle to add a second aminoacid. In certain embodiments, third, fourth, fifth, and subsequent aminoacid addition cycles may be performed to produce an immobilized peptideof any desired length. In some embodiments, at least about 10 amino acidaddition cycles, at least about 50 amino acid addition cycles, or atleast about 100 amino acid addition cycles are performed, resulting inthe addition of at least about 10 amino acid residues, at least about 50amino acid residues, or at least about 100 amino acid residues to theimmobilized peptides. In certain such embodiments, a relatively highpercentage of the amino acid addition cycles (e.g., at least about 50%,at least about 75%, at least about 90%, at least about 95%, or at leastabout 99% of such amino acid addition cycles) can be performed at highyield (e.g., at least about 99%, at least about 99.9%, at least about99.99%, or substantially 100%). In some such embodiments, a relativelyhigh percentage of the amino acid addition cycles (e.g., at least about50%, at least about 75%, at least about 90%, at least about 95%, or atleast about 99% of such amino acid addition cycles) can be performedquickly, for example, within any of the time ranges specified above orelsewhere herein. In some such embodiments, a relatively high percentageof the amino acid addition cycles (e.g., at least about 50%, at leastabout 75%, at least about 90%, at least about 95%, or at least about 99%of such amino acid addition cycles) can be performed with limited or nodouble incorporation, for example, within any of the doubleincorporation ranges specified above or elsewhere herein.

In embodiments in which there are more than one addition cycles, thetotal amount of time that passes between the end of an amino acidaddition cycle and a subsequent amino acid addition cycle may berelatively short. For example, in certain embodiments in whichfluorenylmethyloxycarbonyl protection groups are employed, the totalamount of time between the ends of the first and second amino acidaddition cycles is about 10 minutes or less, about 9 minutes or less,about 8 minutes or less, about 7 minutes or less, about 6 minutes orless, about 5 minutes or less, about 4 minutes or less, about 3 minutesor less, about 2 minutes or less, about 1 minute or less, from about 10seconds to about 10 minutes, from about 10 seconds to about 9 minutes,from about 10 seconds to about 8 minutes, from about 10 seconds to about7 minutes, from about 10 seconds to about 6 minutes, from about 10seconds to about 5 minutes, from about 10 seconds to about 4 minutes,from about 10 seconds to about 3 minutes, from about 10 seconds to about2 minutes, or from about 10 seconds to about 1 minute. In certainembodiments in which protection groups comprising,fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, or any other suitableprotection group are employed, the total amount of time between the endof an amino acid addition cycle and a subsequent amino acid additioncycle may be about 5 minutes or less, about 4 minutes or less, about 3minutes or less, about 2 minutes or less, about 1 minute or less, fromabout 10 seconds to about 5 minutes, from about 10 seconds to about 4minutes, from about 10 seconds to about 3 minutes, from about 10 secondsto about 2 minutes, or from about 10 seconds to about 1 minute.

As mentioned above, certain aspects relate to processes and systems thatallow the total time required for one or more addition cycles to besignificantly reduced compared to previous solid phase peptide synthesismethods. Since the advent of continuous solid phase peptide synthesisover 30 years ago, continual efforts have focused on improving itsutility and applicability. While these improvements have contributed tothe commercial success of automated solid phase peptide synthesizers,reducing synthesis time still remains a significant barrier. Over 30years of research and development in the field have been unable toproduce fast synthesis techniques. Typical continuous solid phasepeptide synthesis using Fmoc or Boc protection groups require 30 to 90minutes to add a single amino acid. Certain processes and techniqueshave been discovered, with the context of the present invention, thataddress the long felt need to decrease synthesis time. For example, fastsynthesis times may be achieved by employing specialized techniques formixing, heating, and/or controlling pressure drop.

Certain steps in the amino acid addition cycle may require mixing ofreagents. In some conventional systems, reagents are mixed a long timebefore exposure to the immobilized peptides, which may result inundesirable side reactions and/or reagent degradation prior to exposureto the immobilized peptides. In some instances, the side reactionsand/or degradation adversely affects the yield and kinetics of step inthe amino acid addition cycle (e.g., amino acid exposing step). In someconventional systems, reagents are mixed in the presence of theimmobilized peptides, which may result, e.g., in slower reactionkinetics. One technique for achieving rapid peptide synthesis mayinvolve merging reagent streams prior to, but within a short amount oftime of, arrival at the immobilized peptides, as shown in FIG. 1.

In some embodiments, a process for adding amino acid residues topeptides comprises flowing a first stream comprising amino acids,flowing a second stream comprising an amino acid activating agent (e.g.,an alkaline liquid, a carbodiimide, and/or a uronium activating agent).For example, referring back to FIG. 1, reagent reservoir 25 may compriseamino acids. Reagent reservoir 26 may comprise, in some suchembodiments, an amino acid activating agent. The first and secondstreams may be merged to form a mixed fluid comprising activated aminoacids. For example, referring to FIG. 1, amino acids from reservoir 25can be flowed in first stream 30, and amino acid activating agent can beflowed in second stream 32. First stream 30 and second stream 32 can bemixed, for example, at point 34 of stream 40. The mixed fluid maycomprise activated amino acids due to the activation of the amino acidsby the amino acid activating agent.

In certain embodiments, after the amino acids have been activated, theimmobilized peptides may be exposed to the mixed fluid within arelatively short period of time. For example, in certain embodiments,the plurality of peptides immobilized on the solid support may beexposed to the mixed fluid within about 30 seconds (or within about 15seconds, within about 10 seconds, within about 5 seconds, within about 3seconds, within about 2 seconds, within about 1 second, within about 0.1seconds, or within about 0.01 seconds) after merging the first andsecond streams to form the mixed fluid.

In certain embodiments, merging reagent streams may be used in an aminoacid addition cycle, as described herein. For example, a first fluidstream comprising amino acids and a second stream comprising an aminoacid activating agent may be merged to form a mixed fluid comprising theactivated amino acids within about 30 seconds prior to exposing theactivated amino acids to peptides immobilized on a solid support. Insome embodiments, in which more than one amino acid addition cycle isperformed, one or more amino acid addition cycles (e.g., a first and asecond amino acid addition cycle) may comprise merging a first fluidstream comprising amino acids and a second stream comprising an aminoacid activating agent to form a mixed fluid comprising activated aminoacids within about 30 seconds prior to exposing the amino acids to thesolid support. It should be understood that merging reagent streams maybe used in connection with any suitable step in the addition cycle andmay be used in connection with one or more steps in an amino acidaddition cycle.

In general, streams may be merged using any suitable technique known tothose of skill in the art. In some embodiments, the streams may bemerged by flowing the first stream and the second stream substantiallysimultaneously into a single stream (e.g., by merging channels throughwhich the streams flow). Other merging methods may also be used.

Another technique for achieving fast synthesis times may involve heatinga stream prior to, but within a short period of time of, arrival at thereactor. Supplying the reactor with a heated stream may alter thekinetics of a process occurring in the reactor. For example, exposingimmobilized peptides, solid supports, or other synthesis components to aheated stream may alter the reaction kinetics and/or diffusion kineticsof the amino acid addition process. For example, exposing the peptidesto a heated stream comprising activated amino acids may increase therate at which amino acids are added to the peptides.

Thus, in some embodiments, a process for adding amino acid residues topeptides may comprise heating a stream comprising activated amino acidssuch that the temperature of the activated amino acids is increased byat least about 1° C. (or at least about 2° C., at least about 5° C., atleast about 10° C., at least about 25° C., at least about 50° C., and/orless than or equal to about 100° C., and/or less than or equal to about75° C.) prior to the heated amino acids being exposed to the immobilizedpeptides. In certain embodiments, a stream comprising any othercomponent (e.g., a washing agent, a deprotection agent, or any othercomponents) may be heated such that the temperature of the streamcontents is increased by at least about 1° C. (or at least about 2° C.,at least about 5° C., at least about 10° C., at least about 25° C., atleast about 50° C., and/or less than or equal to about 100° C., and/orless than or equal to about 75° C.) prior to the stream contents beingexposed to the immobilized peptides. In some instances, the heating step(e.g., the heating of the activated amino acids and/or the heating ofany other component within a stream transported to the immobilizedpeptides) may be performed within about 30 seconds (or within about 15seconds, within about 10 seconds, within about 5 seconds, within about 3seconds, within about 2 seconds, within about 1 second, within about 0.1seconds, or within about 0.01 seconds) of exposing the stream contents(e.g., the heated activated amino acids) to the immobilized peptides. Insome such embodiments, and as illustrated in the exemplary embodiment ofFIG. 1, such heating may be achieved by heating a location upstream ofthe immobilized peptides. In some such embodiments, the heating of theamino acids begins at least about 0.1 seconds, at least about 1 second,at least about 5 seconds, or at least about 10 seconds prior to exposureof the amino acids to the immobilized peptides. In certain embodiments,the amino acids are heated by at least about 1° C. (or at least about 2°C., at least about 5° C., at least about 10° C., at least about 25° C.,at least about 50° C., and/or less than or equal to about 100° C.,and/or less than or equal to about 75° C.) at least about 0.1 seconds,at least about 1 second, at least about 5 seconds, or at least about 10seconds prior to the amino acids being exposed to the immobilizedpeptides.

Referring back to FIG. 1, for example, system 5 may comprise heatingzone 42, within which the contents of stream 40 may be heated. Heatingzone 42 may comprise a heater. In general, any suitable method ofheating may be used to increase the temperature of a stream. Forexample, heating zone 42 may comprise a liquid bath (e.g., a waterbath), a resistive heater, a gas convection-based heating element, orany other suitable heater. In some instances, the heating mechanism maybe within a short distance of the immobilized peptides, for example,within about 5 meters, within about 1 meter, within about 50 cm, orwithin about 10 cm.

In some embodiments, including those illustrated in FIG. 1, both theheating of the amino acids and the merging of the amino acids with theamino acid activating agent (e.g., an alkaline liquid, a carbodiimide,and/or a uronium activating agent) can be performed before and within arelatively short time of the amino acids contacting the immobilizedpeptides. Heating the amino acids may be performed before, during,and/or after merging the stream comprising the amino acids with thestream comprising the amino acid activating agent.

In certain embodiments, heating a stream just prior to being exposed tothe immobilized peptides (as opposed to heating the stream long beforetransport of the stream contents to the immobilized peptides) mayminimize the thermal degradation of one or more reagents (such as, forexample, the amino acids that are to be added to the peptides and/or thedeprotection reagent) in the stream. Of course, as discussed above,heating a stream prior to arrival of the stream components can enhancethe speed with which a reaction or washing step may be performed.

In some embodiments, a heating step may be used in an amino acidaddition cycle, as described herein. For example, heating the activatedamino acids, such that the temperature of the activated amino acids isincreased by at least about 1° C., may be performed prior to and withinabout 30 seconds (or within any of the other time ranges mentionedelsewhere) of exposing the activated amino acids to the immobilizedpeptides. In certain embodiments, in which more than one amino acidaddition cycle is performed, one or more amino acid addition cycles(e.g., a first and a second amino acid addition cycle) may compriseheating the activated amino acids prior to and within about 30 seconds(or within any of the other time ranges mentioned elsewhere) of exposingthe activated amino acids to the immobilized peptides. It should beunderstood that a heating step may be used in connection with anysuitable step in the addition cycle and may be used in connection withone or more steps of any individual addition cycle or with all steps ofa series of addition cycles.

As noted above, in some embodiments, heating a stream may increase thetemperature of the stream contents (e.g., may increase the temperatureof amino acids within the stream) by at least about 1° C., at leastabout 2° C., at least about 5° C., at least about 10° C., at least about25° C., or at least about 50° C. In some instances, heating a stream mayincrease the temperature of the stream contents (e.g., may increase thetemperature of amino acids within the stream) by less than or equal toabout 100° C. or less than or equal to about 75° C. Combinations of theabove-referenced ranges are also possible (e.g., at least about 1° C.and less than or equal to about 100° C., etc.).

Systems and methods for reducing pressure drop across the immobilizedpeptides may be used, according to certain embodiments, to improve thespeed of peptide synthesis. In some embodiments, the flow rate ofreagents across the immobilized peptides may influence the speed ofpeptide synthesis. For example, the time required for one or more stepsin an amino acid addition cycle (e.g., deprotection reagent exposingstep, deprotection reagent removal step, activated amino acid exposingstep, activated amino acid removal step) may decrease with increasingflow rate. In general, the use of high flow rates ensures that theconcentration of reagent near the immobilized peptides is not depletedas severely as might be observed when low flow rates are employed. Inmany traditional continuous solid phase peptide synthesis systems, theflow rate is limited by the pressure drop across the reactor. Pressuredrop may occur due to expansion of the solid support during synthesisand/or due to improper sizing of process equipment. In certainembodiments, the pressure drop across the solid support during an aminoacid addition cycle may not exceed about 700 psi for more than about 5%(or for more than about 1%) of the time period during which the cycle isperformed. For example, in certain embodiments, during each step of anamino acid addition cycle (e.g. the deprotection reagent exposing step,the deprotection reagent removal step, the activated amino acid exposingstep, and the activated amino acid removal step) the pressure dropacross the solid support may not exceed about 700 psi for more thanabout 5% (or for more than about 1%) of the time period during which thestep is performed. In embodiments in which more than one addition cycleis performed, the pressure drop during one or more addition cycles(e.g., the first and second amino acid addition cycle) may not exceedabout 700 psi for more than about 5% (or for more than about 1%) of thetime period during which the cycle is performed.

In some embodiments, the pressure drop across reactor during each stepof an amino acid addition cycle and/or during one or more additioncycles may not exceed about 700 psi, about 600 psi, about 500 psi, about400 psi, about 250 psi, about 100 psi, or about 50 psi for more thanabout 5% (or for more than about 1%) of the time period during which thestep is performed.

In certain embodiments, the pressure drop across reactor may be reducedby using a process vessel (e.g., the column of a packed column) with adesirable aspect ratio. Generally, the aspect ratio of a process vesselis the ratio of the length of the vessel (substantially parallel to thedirection of flow through the vessel) to the shortest width of thevessel (measured perpendicular to the length of the vessel). As anexample, in the case of a cylindrical vessel, the aspect ratio would bethe ratio of the height of the cylinder to the cross-sectional diameterof the cylinder. Referring back to FIG. 1, for example, the aspect ratioof reactor 10 would be the ratio of the length of dimension A to thelength of dimension B (i.e., A:B). In some embodiments, the aspect ratioof the reactor may be less than or equal to about 20:1, less than orequal to about 10:1, less than or equal to about 5:1, less than or equalto about 3:1, less than or equal to about 2:1, less than or equal toabout 1:1, less than or equal to about 0.5:1, less than or equal toabout 0.2:1, or less than or equal to about 0.1:1 (and/or, in certainembodiments, as low as 0.01:1, or lower).

In some embodiments, relatively short addition cycles with high yieldsand/or limited and/or no double incorporation may be achieved byemploying one or more of the techniques described herein. For example,certain of the systems and methods described herein may allow the aminoacid exposing step (i.e., the step of exposing the activated amino acidsto the immobilized peptides) to be performed (e.g., while achieving thehigh yields and/or avoiding double incorporation to any of the degreesdescribed herein) in about 1 minute or less (e.g., about 30 seconds orless, about 15 seconds or less, about 10 seconds or less, about 7seconds or less, or about 5 seconds or less, and/or, in certainembodiments, in as little as 1 second, or less). In some instances,certain of the systems and methods described herein may allow thedeprotection reagent removal step and/or the activated amino acidremoval step to be performed in about 2 minutes or less (e.g., about 1.5minutes or less, about 1 minute or less, about 45 seconds or less, about30 seconds or less, about 15 seconds or less, about 10 seconds or less,about 5 seconds or less, and/or, in certain embodiments, in as little as1 second, or less). In certain embodiments, certain of the systems andmethods described herein may allow the deprotection reagent exposingstep (i.e., the step of exposing the immobilized peptides to thedeprotection reagent) to be performed in about 20 seconds or less (e.g.,about 15 seconds or less, about 10 seconds or less, about 8 seconds orless, about 5 seconds or less, about 1 second or less, and/or, incertain embodiments, in as little as 0.5 seconds, or less).

In certain cases, the time required for peptide synthesis may beinfluenced by the choice of protection group. For example, the use ofFmoc protection groups is generally understood to require longersynthesis cycle times. However, the systems and methods described hereincan be used to perform fast amino acid addition, even when Fmocprotection group chemistries are employed. In some embodiments, thetotal time for an amino acid addition cycle may be low, regardless ofthe type of protection group that is being used.

In general, any protection group known to those of ordinary skill in theart can be used. Non-limiting examples of protection groups (e.g.,n-terminal protection groups) include fluorenylmethyloxycarbonyl,tert-butyloxycarbonyl, allyloxycarbonyl (alloc), carboxybenzyl, andphotolabile protection groups. In certain embodiments, immobilizedpeptides comprise fluorenylmethyloxycarbonyl protection groups. In someembodiments, immobilized peptides comprise tert-butyloxycarbonylprotection groups.

As described elsewhere, an amino acid activating agent may be used toactivate or complete the activation of amino acids prior to exposing theamino acids to immobilized peptides. Any suitable amino acid activatingagent may be used. In certain embodiments, the amino acid activatingagent comprises an alkaline liquid. The amino acid activating agentcomprises, in some embodiments, a carbodiimide, such asN,N′-dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and the like. Incertain embodiments, the amino acid activating agent comprises a uroniumactivating agent, such asO-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HBTU); 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU);1-[(1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholino)]uronium hexafluorophosphate (COMU); and thelike.

As described elsewhere, peptides may be immobilized on a solid support.In general, any solid support may be used with any of the additioncycles described herein. Non-limiting examples of solid supportmaterials include polystyrene (e.g., in resin form such as microporouspolystyrene resin, mesoporous polystyrene resin, macroporous polystyreneresin), glass, polysaccharides (e.g., cellulose, agarose),polyacrylamide resins, polyethylene glycol, or copolymer resins (e.g.,comprising polyethylene glycol, polystyrene, etc.).

The solid support may have any suitable form factor. For example, thesolid support can be in the form of beads, particles, fibers, or in anyother suitable form factor. In some embodiments, the solid support maybe porous. For example, in some embodiments macroporous materials (e.g.,macroporous polystyrene resins), mesoporous materials, and/ormicroporous materials (e.g., microporous polystyrene resin) may beemployed as a solid support. The terms “macroporous,” “mesoporous,” and“microporous,” when used in relation to solid supports for peptidesynthesis, are known to those of ordinary skill in the art and are usedherein in consistent fashion with their description in the InternationalUnion of Pure and Applied Chemistry (IUPAC) Compendium of ChemicalTerminology, Version 2.3.2, Aug. 19, 2012 (informally known as the “GoldBook”). Generally, microporous materials include those having pores withcross-sectional diameters of less than about 2 nanometers. Mesoporousmaterials include those having pores with cross-sectional diameters offrom about 2 nanometers to about 50 nanometers. Macroporous materialsinclude those having pores with cross-sectional diameters of greaterthan about 50 nanometers and as large as 1 micrometer.

One advantage of the inventive systems and methods described herein isthat they can be used with standard solid support materials withoutdegradation in performance. For example, in certain embodiments, astandard commercial polystyrene resin support can be used. In manyprevious systems, such supports collapsed when used in flow-based solidphase peptide synthesis systems, causing an increase in pressure drop.As the resin swells during synthesis, it becomes increasingly likely tocollapse, which causes an increase in the pressure drop across theresin, requiring an increase in applied pressure to maintain a constantflow rate. The increase in applied pressure can lead to more severecollapse of the resin, leading to a positive feedback effect in whichthe pressure applied to the fluid must be repeatedly increased. Atsufficiently high pressures, the resin may extrude through any frit orother system used to confine it. The systems and methods describedherein can be used to manage pressure drop such that the resin(including standard polystyrene resins and other standard resins) do notcollapse during synthesis or collapse only to a degree that does notresult in the positive feedback effect described above, leading to amore stable and controllable system. In certain embodiments, the solidsupport is contained within a packed column.

As used herein, the term “peptide” has its ordinary meaning in the artand may refer to amides derived from two or more amino carboxylic acidmolecules (the same or different) by formation of a covalent bond fromthe carbonyl carbon of one to the nitrogen atom of another with formalloss of water. An “amino acid residue” also has its ordinary meaning inthe art and refers to the composition of an amino acid (either as asingle amino acid or as part of a peptide) after it has combined with apeptide, another amino acid, or an amino acid residue. Generally, whenan amino acid combines with another amino acid or amino acid residue,water is removed, and what remains of the amino acid is called an aminoacid residue. The term “amino acid” also has its ordinary meaning in theart and may include proteogenic and non-proteogenic amino acids.

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

Example 1

This example describes a flow based platform for rapid Fmoc solid phasepeptide synthesis, in which an amino acid was incorporated in steps ofunder five minutes. In this example, each step for amino acid addition(e.g., amide bond formation, wash, and N-termini deprotection) wascarried out under a constant stream of fluid passed over a resinconfined in a small, fritted plastic tube. Flow methods, as opposed tocommonly used batch methods, allowed for the consistent rapidpreheating, addition, and removal of solvents and reagents. Theconsistent rapid preheating, addition, and removal of solvents andreagents allowed a 5 minute cycle time, which included a 30 secondamide-bond formation step. A number of model peptides were prepared,without double coupling or double deprotection. In addition, good yieldsand high purity, as shown by liquid chromatography-mass spectrometry(LC-MS), were achieved. This approach was also applied to the synthesisof a 58-residue protein from three polypeptide segments. The longestfragment, a 27 residue peptide, was prepared in 2.3 hours, which was 10fold faster than conventional Fmoc methods. It is believed thatautomating various processing steps, increasing flow rate, reducingunnecessarily long wash times, and using a smaller aspect ratio reactorwould substantially reduce the synthesis times reported here.

As shown in FIG. 2A, a high pressure liquid chromatography (HPLC) pumpwas used to deliver either a piperidine deprotection solution or adimethylformamide (DMF) wash solvent to the reaction vessel. A manuallyactuated 3-way valve was used to select which reagent was delivered tothe reaction vessel. The HPLC pump outlet was attached to the reactionvessel via a luerlock quick connect. For the coupling step, the quickconnect was manually moved to a syringe pump, which delivered a solutionof activated amino acid. It is believed that even faster performancethan that reported here could be achieved by automating this step. Thereactor effluent was passed through a UV detector to continuouslymonitor the absorbance at 304 nm, a region where Fmoc amino acids absorbstrongly. The reactor was designed to be simple and easy to construct. A¼″ inner diameter by 3.5″ long perfluoroalkoxy tube with Swagelokreducing unions as the inlet and outlet was used. A frit was positionedin the outlet using a short piece of tubing with a ¼ in. outer diameter.Installation of the outlet fitting and concurrent compression of theferrule and tube sealed the frit in place as seen in FIG. 2B. The totalvolume of the reactor was about 2.5 mL. This design held up to 100 mg ofresin and was used to prepare peptides up to 27 residues in length.

To verify the feasibility of Fmoc SPPS with the flow based SPPS system,the model peptide Fmoc-ALFALFA-CONH2 was synthesized on a 0.1 mmol scaleusing 100 mg of resin. Based on an initial estimate, a 2 minute DMF washat 10 mL/min, a 2 minute Fmoc deprotection step at 6 mL/min, and anotherDMF wash, and a 6 minute room temperature coupling with activated aminoacid delivered at 1 mL/min were chosen as the starting point for anamino acid addition cycle. This sequence allowed efficient peptidesynthesis in 12 minutes per residue. The reverse phase (RP)-HPLC tracefor the crude peptide is shown in FIG. 2C.

After validating this approach, improved wash step, Fmoc removal, andcoupling times were determined. All subsequent studies were carried outat 60° C. to reduce the cycle time without significantly increasingformation of side products. The final synthetic timeline, which was usedin all subsequent experiments, is shown in FIG. 3C. The final synthetictimeline has a 2 minute DMF wash at 10 mL/min, a 20 second Fmocdeprotection step at 10 mL/min, another 2 minute DMF wash, and a 30second coupling step with activated amino acid delivered at 12 mL/min.This approach was studied by synthesizing the peptide ACP(65-74). Thispeptide served as a model to validate the flow based SPPS platform,because ACP(65-74) was considered difficult to prepare. It is believedthat substantial reductions in synthesis times could be achieved whensynthesizing peptides that are easier to prepare.

In conventional systems, the main synthetic impurity in the synthesis ofACP(65-74) is a chromatographically resolved Val deletion. The LCMS datafor the synthesis of ACP(65-74) with the flow based SPPS platformmethodology, as well as two controls, is shown in FIGS. 4A-4D. Using theadjusted protocol and the HATU coupling agent, a minor Val deletionproduct was observed. When using HBTU, more Val deletion was observed,which is consistent with prior reports. ACP(65-74) synthesized with theflow based SPPS platform, but at room temperature, showed large Val andGln deletions, confirming that temperature is important. No majordifferences between the product composition from the room temperaturesynthesis and an analogous batch synthesis were observed. Two additional“difficult” peptides, a conotoxin variant and a fragment of the HIV-1protease, were also synthesized. The LCMS data is shown in FIGS. 5A-5B.Both of these peptides contained cysteine residues that were observed toracemize during activation. Therefore, model studies using the peptideGCF were carried out. Using the model study, several conditions thatproduced less than 1% diastereomer, as shown in FIGS. 6A-6E, were found.This level of racemization is consistent with literature for Fmocprotocols.

Using the modified coupling conditions for cysteine, the conotoxinvariant and a fragment of the HIV-1 protease peptides were prepared on a0.1 mmol scale. Eighty nine milligrams (53%) of the crude conotoxin and90 mg (43%) of the crude HIV-I protease fragment were isolated. Toexplore the utility of the flow platform in the preparation of syntheticproteins, a 58 residue tri-helical protein based on the Z domain ofprotein A (referred to as the affibody) was prepared. The syntheticstrategy, which can be seen in FIG. 7A, used peptide-hydrazides asthioester precursors for use in native chemical ligation. Peptidehydrazides can be oxidized with NaNO₂ to form a C-terminalpeptide-azide, which can react with a thiol to form a peptide thioester.The LCMS data for the crude synthetic peptides are shown in FIGS. 7B-D.Variants of these peptides were also prepared using Boc in-situneutralization methods and the peptides were found to be of similarcrude quality (FIGS. 8A-8F). Retention time shifts are due to differentchromatographic conditions. Each peptide for the affibody was purified(FIG. 9), the affibody was then synthesized, and the highly pure,full-length affibody was isolated after purification (FIG. 7E).

Although it was possible to implement this protocol in a batch mode, theflow based platform overcame a number of significant obstacles. First,the completely sealed reactor and a preheat loop were immersed in atemperature controlled bath which allowed reagents to be heated in aconsistent and controlled manner immediately before reaching the resinbed. This would be difficult in some batch systems. Second, the use of alow volume reactor (about 2.5 mL) and narrow tubing for delivery ofsolvents and reagents allowed efficient washing with only 20 mL ofsolvent. In contrast, batch mode automated and manual synthesestypically use large volumes of solvent (about 70 mL per wash). Third,the flow platform was assembled from common laboratory equipment at lowcost without machine or glass shop support. Fourth, high qualitypeptides were obtained quickly without double coupling, doubledeprotection, ninhydrin or Kaiser tests, or resin mixing. During thestudies with ACP(65-74), no decrease in the Val deletion peptide wasobserved after double coupling Val and double deprotecting the precedingGln. These additional steps are often employed in batch mode syntheses.Finally, the flow based SPPS system was capable of being adapted tolarger synthetic scales by increasing the diameter of reactor. Forexample, the reactor diameter was doubled and the resulting reactor usedto synthesize ACP(65-74) on a 0.2 mmol scale using exactly the sameprotocol. Another option for increasing the synthetic scale was tosimply increase the reactor length. However, this strategy significantlyincreased the backpressure, which may pose difficulties duringsynthesis. The flow based SPPS platform in this example allowed for therapid Fmoc synthesis of polypeptides. It was found that, under flow at60° C., amide-bond formation and Fmoc removal were fast (within seconds)and did not improve with increased reaction time. Using the flow basedFmoc system, three affibody segments were able to be synthesized andcleaved in one working day. By contrast, the production of similarpeptides using optimized Boc in-situ neutralization methods, with 15minute cycle times, required more than three days. In addition, thepurified peptides were ligated to generate synthetic proteins. Thisapproach allowed for the rapid production of highly pure, moderatelysized peptides that were easily ligated to obtain larger fragments.

Example 2

This example describes the determination of the deprotection step time.Real-time monitoring of the effluent with an inline UV-Vis detectorallowed the deprotection step to be reduced in length. The rate of Fmocremoval was investigated by monitoring the UV absorbance of the reactoreffluent at 304 nm. To determine the minimum treatment time for robustFmoc removal, the deprotection solution was flowed in at 10 mL/min for60 seconds, 30 second, 15 seconds, or 6 seconds. Twenty seconds at 10mL/min was found to be sufficient for complete Fmoc removal. EffectiveFmoc removal was also achieved during the 6 second steps.

In developing an Na deprotection protocol, piperidine in DMF wasselected as the standard deblocking reagent. A concentration of 50%(v/v) in DMF was selected over the more common 20% (v/v) in DMF becausethe deprotection solution was diluted as it entered the column. A higherconcentration was therefore desirable. The flow rate was set at 10mL/min (maximum) to reach an effective concentration in the minimumtime. To determine the length of the deprotection step, ALF peptide wassynthesized with a double deprotection of every residue, and the UVabsorbance of the effluent was monitored at 304 nm. Piperidine and DMFdid not absorb well at this wavelength, but piperidine-DBF, thedeprotection product, did. Therefore, the presence of a second peakafter the second deprotection indicated that the initial deprotectionwas inadequate. No second peak was observed after 60 seconds, 30seconds, and 15 seconds of deprotection, and only a very small peak wasobserved after a 6 second initial deprotection. In all cases, the firstdeprotection was at 10 mL/min, and the second was for one minute at 10mL/min. Since, Fmoc removal has been reported to be sequence dependent,a final deprotection time of 20 seconds was selected. However, it isbelieved that the 6 second deprotection step (and even fasterdeprotection steps) would be suitable for many peptide synthesisprocesses. Additionally, it is believed that, by increasing the flowrate of the deprotection agent, robust Fmoc removal can be achieved inone second or less.

The double deprotection protocol had to be used to determinedeprotection time because it took significantly longer to wash thepiperidine-DBF adduct out of the resin than to remove the Na Fmoc group.If the effluent was simply monitored until the absorbance returned tonear-baseline, most of the “deprotection” time would have been spentwashing the resin with deprotection reagent after the deprotection wascomplete.

FIG. 10 shows the UV record of the incorporation of the final eightresidues during the conotoxin synthesis. Negative marks represent manualactions. The scanned trace has been color enhanced and a time-lineadded, taking zero to be the beginning of the trace. The marks of onecycle have been annotated with 1 indicating the end of the previouswash, 2 indicating the beginning of coupling, 3 indicating the end ofcoupling, 4 indicating the start of the first wash, 5 indicating the endof the first wash and start of the deprotection, and 6 indicating theend of the deprotection and start of the second wash. The quick connectwas moved between 1 and 2, and between 3 and 4. Inconsistencies in cycletime and missing marks were due to human error.

Example 3

This example describes the determination of the wash step time.Real-time monitoring of the effluent with an inline UV-Vis detectorallowed the wash step to be reduced in time. The efficiency of the washstep was systematically investigated by monitoring the UV absorbance ofthe reactor effluent at 304 nm. The time required to wash the amino acidout of the reactor as a function of flow-rate was then investigated. Itwas determined that the wash efficiency was principally determined bythe total volume of solvent used, with about 16 mL of DMF required toremove 99% of the amino acid precursor. However, at flow rates greaterthan about 6 mL/min, marginally less solvent was required. It wasconcluded that a 2 minute DMF wash at 10 mL/min was sufficient. Doubleincorporation of amino acids, which would occur if the DMF wash did notcompletely remove the amino acid or deprotection solution, was notobserved for the 2 minute wash time. Increasing the wash volume did notimprove the crude peptide quality. It is believed that even faster washtimes could be observed by, for example, increasing flow rate, changingthe geometry of the inlet to reduce recirculation, and/or reducing theaspect ratio of the reactor.

Visual observation of the reactor during wash cycles showedrecirculation and mixing of DMF wash solvent and coupling solution.Other solvent exchanges showed the same behavior. Differences in colorand refractive indices allowed the direct observation of all exchanges.Based on these observations, it was expected that the wash efficiencywas primarily dependent on the volume of solvent used, as predicted by acontinuous dilution model. To test this theory, the UV absorbance of thereactor effluent was monitored at 304 nm during the triplicate synthesisof ACP(65-74). During each synthesis, two consecutive residues werewashed at 10 mL/min, two at 7 mL/min, two at 4 mL/min, two at 2 mL/min,and the final two at 1 mL/min. Wash rates were randomly assigned toblocks of amino acids, ensuring that no block was washed at the samerate twice. The time required for the detector to desaturate wasmeasured for each residue. Desaturation represents approximately 99%reduction in amino acid concentration. This wash efficiency was selectedbecause the wash was essentially complete, but air and particulatecontamination in the detector were less significant than at lower signallevels. The data are shown in FIG. 11. The exponent (parameter b) wassignificantly below negative one. The value of the exponent meant thatless solvent was required to desaturate the detector at higher flowrates. The relationship between desaturation and flow rate was notconsistent with the proposed continuous dilution model.

Based on these results, a maximum flow rate (10 mL/min) was selected forthe wash. The wash time was set at two minutes, which reliably reducedthe final concentration of coupling solution to 0.2% of the initialconcentration. This trend is valid with wash rates up to at least 100ml/min resulting in effective washing observed in 10 seconds. It isbelieved that even faster washing times could be achieved, for example,by increasing the wash liquid flow rate. No double incorporation, theexpected outcome of an inadequate wash, was observed. The apparatus useddid not provide a direct way to monitor the removal of piperidine (theUV absorbance is similar to DMF at accessible wavelengths), so the samewash cycle was used for the second wash. If it was assumed thatpiperidine is removed at the same rate as piperidine-DVB, the two minutewash was an overestimate of the necessary wash time as shown by the UVtrace in FIG. 10. The total time of an amino acid addition cycle may bereduced by substantially reducing the washing step.

Example 4

This example describes the determination of minimum coupling time. Theeffect of coupling time was investigated by synthesizing two modelpeptides: LYRAG-CONH2 and Fmoc-ALF-CONH2. For each of five amino acidaddition cycles, every amino acid was coupled for a nominal time of 90seconds, 45 seconds, 30 seconds, 15 seconds, or 7 seconds at 60° C. asshown in FIG. 3. For LYRAG-CONH2, a significant increase in the Argdeletion peptide was observed when all residues were coupled for 7seconds. For Fmoc-ALF-CONH2, no significant difference in the quality ofthe crude product as a function of coupling time was found. Based onthese results, a 30 second coupling time was concluded to be sufficient.

It is generally known from the literature that, at room temperature,amide-bond formation is 99% complete in less than 100 seconds using HBTUas a coupling agent. If it was assumed that the reaction rate for thisprocess doubled for every 10° C. increase in temperature, at 60° C.amide bond formation would be completed in about 6 seconds, which wouldhave significantly decreased amino acid addition cycle time. Thus, allsubsequent coupling studies were carried out at 60° C. to minimize thecycle time without significantly increasing formation of side products.An important feature of this platform was the ability to simply placethe reactor and a preheat loop in a temperature controlled water bath.The preheat loop allowed reagents to be stored at room temperature andthen immediately heated before entering the reactor, which allowed thethermal degradation of reagents to be minimized.

LYRAG was selected as a model peptide to determine the minimum couplingtime, because the arginine deletion could be monitored. For 90 seconds,45 seconds, and 30 seconds nominal coupling times, the coupling solutionwas delivered at 4, 8, and 12 mL/min, respectively. This flow rateallowed the delivery of 2 mmol of amino acid. Flow rates above 12 mL/minwere not reliably obtainable in this system (although other systemscould be designed to include higher flow rates), so for the 15 secondtrial, half of the coupling solution was used (1 mmol amino acid in 2.5mL 0.4M HBTU in DMF, with 0.5 mL N,N-Diisopropylethylamine (DIEA)). At acoupling time of 7 seconds, the time spent manually moving the quickconnect (5-6 seconds) was very significant, so 1.2 mL of couplingsolution was delivered. This volume was the volume of the preheat loop,so the coupling solution did not reach the reactor until it was clearedfrom the lines by the DMF wash. The wash, at 10 mL/min, took 7.2 secondsto clear 1.2 mL, giving a 7 second coupling time. In the other runs, the5 seconds to move the quick connect was added to the nominal couplingtime, as was the time required to deliver about a 10% increase over thenominal volume of coupling solution. The difference in the time takenfor the DMF wash solvent and coupling solution to clear the inlet linewas subtracted. More accurate coupling times were 93 seconds, 53seconds, 39 seconds, 23 seconds, and 7 seconds. This does not includethe time required to wash the coupling solution from the reactor. Theseven second coupling showed increased Arginine deletion, so the 30second protocol was selected as a conservative estimate. Fmoc-ALF wasproduced with the same procedure, and showed no change in peptidequality with reduction in coupling time. Data are presented in FIG. 3.

It is believed that using an automated system, using higher flow rates,and using a higher temperature would substantially reduce the couplingtime.

Example 5

This example describes the minization of cysteine racemization. Thepeptides Pn1A (A10L) conotoxin, HIV-1 PR(81-99), and GCF were used toexplore techniques to minimize cysteine racemization.

In the initial syntheses of Pn1A (A10L) conotoxin and HIV-1 PR(81-99),cysteine was activated like all other amino acids (1 eq HBTU, 2.9 eqDIEA), and significant diastereotopic impurities were observed in theproducts. These were determined to be the result of cysteineracemization. To investigate conditions that reduce racemization, amodel system, GCF, was selected because the diasteromer formed uponracemization was resolved by RP-HPLC. The standard synthetic procedurewas used, except coupling time was increased to one minute for 60° C.runs and 6 minutes for room temperature (RT) runs. Rink, Gly, and Phewere all activated according to the standard procedure, with oneequivalent of HBTU (5 mL, 0.4M) and 2.9 equivalents of DIEA (1 mL). Forcysteine, various activation procedures were used as summarized below.For procedures that used less than 1 mL of DIEA, DMF was used to replacethis volume. In addition to the activation methods below, an authenticdiasteromer was produced using Fmoc-D-Cys(Trt)-OH and the activationprocedure of 5. TIC traces are shown for 4, 5, 6, 7 and the authenticdiastereomer in FIG. 6. Runs not shown were visually indistinguishablefrom 4. In all cases, the activator, additive, and 2 mmol amino acidwere dissolved in 5 mL DMF, and additional DMF was added as needed. Thebase was added immediately before use. Reaction 8 employed an isolatedC-terminal pentafluorophenyl (Pfp) ester (Fmoc-Cys(Trt)-OPfp) withoutadditional activators, additives, or base. Table 1 summarizes theresults, with racemization quantified by integration of the extractedion current. This enables quantification of racemization below the TICbaseline. The results obtained were consistent with previous reports.

TABLE 1 Reaction conditions and racemization results. Reac- Racemi- tionActivator Additive Base Temp zation 1 HBTU None DIEA 60° C. 10% (1 eq)(2.9 eq) 2 HBTU HOBt DIEA 60° C. 18% (1 eq) (1 eq) (2.9 eq) 3 HBTU NoneDIEA Cys room temp 11% (1 eq) (2.9 eq) G and F 60° C. 4 HBTU None DIEARoom 10% (1 eq) (2.9 eq) temperature 5 HBTU None DIEA 60° C. 1% (1 eq)(0.9 eq) 6 HBTU HOBt DIEA 60° C. 1% (1 eq) (1 eq) (0.9 eq) 7 DCC HOBtNone 60° C. 1% (0.9 eq) (1.1 eq) 8 OPfp None None 60° C. 1%

FIGS. 6A-6E shows GCF produced with various cysteine activation schemes.The peak eluting between the desired product and diastereomer in A and Bwas hydrolysis of the C-terminal carboxamide. The diastereomer wasbarely visible. Conditions are listed in table 1. The conditions in FIG.6A-D show chromatograms for reactions (a) 5, (b) 7, (c) 8, (d) 4, andthe (e) the authentic Gly-D-Cys-L-Phe. The total ion current isdisplayed in each chromatogram.

Example 6

This example describes the synthesis of an affibody. Throughout thissection, ligation buffer refers to a 6M GnHC1, 0.2M Sodium Phosphatebuffer at the specified pH; buffer P is a 20 mM Tris, 150 mM NaClsolution at pH=7.5.

Oxidation chemistry was used to ligate three fragments into a synthetic,58 residue protein. Thiozolidine was found to be unstable in theconditions used, so 9.6 mg of fragment Thz-[28-39]-CONHNH₂ was convertedto a free N-terminal cysteine by treatment with 83 mg methoxyaminehydrochloride in 5 mL of ligation buffer at pH=4 overnight. Quantitativeconversion was observed. The N-to-C assembly shown in FIG. 5A wasemployed instead of the C-to-N synthesis used when thioesters areaccessed directly. Fragment [1-27]-CONHNH₂ was oxidized to theC-terminal azide by dropwise addition of 0.1 mL of 200 mM aqueous NaNO₂to a solution of 11 mg purified fragment [1-27]CONHNH₂ in 1 mL ligationbuffer at pH=3 and 0° C. The reaction proceeded for 20 minutes at 0° C.,and was then quenched by the addition of 172 mg 4-mercaptophenylaceticacid (MPAA) and 34 mg tris(2-carboxyethyl)phosphine•HCl (TCEP•HCl)dissolved in 4.4 mL ligation buffer (pH=7, room temperature (RT)). Tothe resulting thioester, 3.4 mL of the crude methoxyamine treatedfragment Thz-[28-39]-CONHNH₂ were added. After a two hour RT ligation,one half of the crude reaction mixture was purified by RP-HPLC, with 2mg of highly pure material recovered. Of this, 0.6 mg were oxidized bydissolution in 0.1 mL of ligation buffer and dropwise addition of 0.01mL of 200 mM aqueous NaNO₂ at 0° C. The reaction proceeded for 26minutes, and was then quenched by addition of 3.1 mg MPAA and 0.78 mgTCEP•HCl dissolved in 0.1 mL ligation buffer (pH=7, RT). The pH wasadjusted to 7 and 0.3 mg of fragment Cys-[40-58]-CONH₂ were added to thereaction mixture. After a two hour RT ligation, the mixture was dilutedwith 0.21 mL buffer P, then a further 0.63 mL buffer P to fold theresulting affibody. The crude mixture was concentrated over a 3 kDamembrane to a final volume of 0.075 mL. The crude, folded protein wasdiluted with 36 mg TCEP•HCl in 3 mL buffer P and purified to homogeneity(FIG. 9).

FIGS. 9A-9E show LC-MS chromatograms of the purified affibody synthesisintermediates and final product. FIGS. 9A-9E are chromatograms of (a)fragment [1-27]CONHNH₂, (b) fragment Thz-[28-39]-CONHNH₂, (c) fragmentCys-[40-58]-CONH₂, (d) ligation fragment [1-39]-CONHNH₂, and (e) thefinal affibody.

Comparative Example 6

This example describes the synthesis of an affibody using a conventionalmanual Boc in-situ neutralization methods.

Comparable affibody fragments using manual Boc in-situ neutralizationmethods were synthesized. LC-MS data for these crude peptides is shownin FIGS. 8A-8F, next to the crude peptides synthesized on the flow basedSPPS. In all cases, the quality was comparable. Retention time shiftsare due to a change in chromatographic conditions.

Example 7

This example describes the design of the system used to for flow basedSPPS. Throughout this example the system is referred to as thesynthesizer. A schematic of the synthesizer is shown in FIG. 2A. An HPLCpump was used to deliver either methanol purge solvent for washing thepump heads, reactor, and UV detector after use, DMF wash solvent forremoval of reagents and byproducts during synthesis; or 50% (v/v)piperidine in DMF for deprotection of the N-terminus. The positions ofvalves 1 and 2 determined which fluid was delivered. A syringe pump wasused to deliver coupling solution. To switch between the syringe pumpand HPLC pump, a quick connect was manually moved from the outlet of theHPLC pump to the syringe on the syringe pump. A valve would have beengenerally ineffective because the line between the syringe pump andvalve would retain coupling solution, causing incorrect incorporation inthe next cycle. The column and a 1.2 mL preheat loop (not shown) weresubmerged in a water bath to maintain a constant 60° C. Valves 3 and 4selected a high pressure bypass loop used to clear the UV detector whenit was clogged with precipitates, such as the urea byproduct of DCCactivation encountered during cysteine racemization studies. The loopwas also used to purge the detector without the column in line.

A Varian Prostar 210 HPLC pump, KD Scientific KDS200 syringe pump,Varian Prostar 320 UV detector set to 304 nm, Amersham Pharmacia Biotechchart recorder, and VWR 39032-214 water bath were used in thesynthesizer. The HPLC pump delivered about 95% of the nominal flow rate.Disposable 10 mL syringes (BD 309604) were used to deliver couplingsolutions. Valve 1 was a Swagelok ⅛″ 3-way valve (S5-41GXS2). The othervalves in the system were Swagelok 1/16″ 3-way valves (SS-41GXS1). Themethanol, DMF, and 50% (v/v) piperidine lines through valve 1 up tovalve 2 were ⅛″ OD, 1/16″ ID FEP (Idex 1521). The line between valve 3and 4 was 1/16″ OD, 0.010″ ID peek (Idex 1531). All other lines were1/16″ OD, 0.030″ ID PFA (Idex 1514L). To attach the ⅛″ wash anddeprotect lines to the 1/16″ inlets of valve 2, Swagelok ⅛″ to 1/16″(SS-200-6-1) reducing unions were used, followed by a short section of1/16″ tubing. All lengths were minimal, except the tubing between thequick connect and the reactor. This included a 2.6 m (1.2 mL) coil whichwas submerged along with the reactor and served as a preheating loop toensure that reactants were at 60° C. before reaching the reactor.Whenever tubing had to be joined, Swagelok 1/16″ unions were used(SS-100-6). These were used to attach the preheat loop, join the outletof the column to a line from valve 4, and repair a severed bypass loop.The manually changed quick connect was a female luer to 10-32 femaleHPLC fitting (Idex P-659). This connected directly to the syringes onthe syringe pump or to a mating male luer to 10-32 female fitting on theline from valve 3 (Idex P-656). The connection between the UV detectorand chart recorder was a data link (three 18 ga insulated copper wires).

FIG. 2B shows the reactor assembly. The reactor consisted of a tube withstandard compression fittings on each end (⅜″ to 1/16″ reducing unions).On the downstream end there was also a frit. This was positioned by asupport designed to fit inside the reactor and seat against the bottomof the fitting on that end. Various frit porosities were used. The partnumber below was for a 20 micron frit, the most commonly used. The bodywas a 3.5 inch segment of PFA tubing with outer diameter ⅜″ and innerdiameter ¼″. The frit was a ¼″ sintered stainless steel disk 1/16″thick. The frit support was a 0.5″ length of ¼″ OD PTFE tubing. As thefittings were tightened, the nut compressed the ferrule against thefitting body, sealing the reactor body to the fitting body. This alsocompressed the reactor body against the frit, forming an internal sealagainst the frit. The reactor body and frit were purchased fromMcMaster-Carr as part numbers S1805K73 and 94461314, respectively. Thenut, ferrule, and fitting body are available as a set with the 1/16″ nutand ferrule from Swagelok as part number SS-600-6-1. Replacementferrules are available as SS-600-SET. The reactor was assembled by firstcutting the body and frit support to length, ensuring the ends weresquare. A sharp razor blade and steady hand were used for theseoperations. Next, the outlet (downstream) end was assembled. The fritwas placed on a solid, clean surface and the reactor body was pressedonto it. After verifying that the frit was square and flush with the endof the reactor, the frit support was pushed in slightly, pushing thefrit up towards its final position. Firmly seating the reactor body inthe fitting body forced the frit to its final position. It was verifiedthat the frit was square and properly positioned under the ferrule, thenthe fitting was installed according to the manufacturer's instructions.Once sealed, the frit could not be removed and reseated. Finally, theinlet fitting was installed according to the manufacturer'sinstructions. A high pressure reactor with a stainless steel body wasalso built. In this case, the downstream fitting had to be tightenedwell beyond specification to effect a seal with the frit. The reactorwas typically replaced every 3-8 syntheses. When replacing the reactor,the ferrules, frit and reactor body were not reused. All other partswere reused. The nuts were recovered by cutting the reactor body inhalf.

To load the reactor, the upstream fitting body was removed and a slurryof resin in methanol was pipetted in. The reactor was completely filledwith methanol, and the fitting body was reinstalled. The inlet line andpreheat loop were filled with solvent by attaching them to the quickconnect and running the HPLC pump before attaching them to the reactor.The reactor was then kept upright in the water bath so that any smallbubble would move to the top and not interfere with wetting the resin.Before the first coupling, the resin was washed for two minutes with DMFat 10 ml/min.

Example 8

This example describes the design of a large scale reactor for use inflow based SPPS. FIG. 12 shows the larger reactor. The design principlesof the small scale reactor used for all syntheses translated directly tolarger scales. In order to preserve the same cycles, however, the volumeof the reactor had to be constant. Two problems were encountered whenscaling up four times to a ⅝″ OD, ½″ ID tube. First, there were nostandard ⅝″ to 1/16″ compression fittings. Second, the minimum distancebetween ⅝″ fittings is quite large, meaning there is a large minimumvolume. To overcome the first problem, a ⅝″ to ⅜″ fitting followed by a⅜″ to 1/16″ fitting was used, but this necessitated a joining length of⅜″ tubing that greatly increased the already large volume of thereactor.

To reduce the reactor volume, a 316SS insert was machined that consistedof a nominal ½″ OD segment followed by a ⅜″ OD segment with a ¼″ throughhole. A ⅝″ to ⅜″ reducing union was bored out to give a ⅜″ through hole,the insert was seated, and the ⅜″ ferrule swaged on. After this, theinsert could not be separated from the fitting. When installed, the ½″part of this insert-fitting sat in top of the reactor and limited thevolume.

This was effective, but there was still a large volume from the ¼″ hole.This volume was reduced by inserting a ¼″ OD, ⅛″ ID PFA tube and cuttingit flush. To further reduce the volume, a ⅛″ OD, 1/16″ ID PFA tube wasinserted by heating and drawing a section of tubing to a narrowerdiameter, threading it through, and pulling until all tubing in theinsert was of the proper diameter. Both sides of the tube were cut flushand the drawn section was discarded. A ⅜″ to 1/16″ reducing union wasinstalled on the open end of the ⅜″ segment to interface with the restof the system. This insert-fitting is pictured in FIG. 12A (left). Toprevent the upstream insert fitting from becoming permanently sealedinto the tube like the frit, the nominal ½″ segment was machined to0.496″ and polished.

A similar piece was machined for the outlet side, with a ½″ section theproper length to seat the frit under the ferrule. To prevent all of thesolvent from being forced through a small central section of the frit, a⅜″ step 0.05″ deep was cut. The bottom of this hole tapered to a ⅛″through hole at 31 degrees from horizontal (a standard drill bit taper).A ⅛″ OD, 1/16″ ID PFA tube was inserted to further limit the volume.This piece positioned the frit and sat largely below the ferrule, so astandard finish was adequate. The one pictured in FIG. 12A is PTFE, andwas installed in a bored through ⅝″ to ⅜″ reducing union in exactly thesame way as the upstream insert. A ⅜″ to 1/16″ reducing union wasinstalled on the open end of the ⅜″ segment to interface with the restof the system.

Tubing was used to limit the internal volume, rather than directlymaking a small hole, to simplify fabrication.

To assemble the reactor, the frit was pressed in and the downstreaminsert-fitting installed as a regular fitting. The upstreaminsert-fitting was then installed as a regular fitting. Despite cuttingit undersize and polishing, the upstream inset-fitting was very tightand difficult to remove. For subsequent reactors, an aluminum spacer wasused so that the nut could not move down, and would instead eject theinsert-fitting when loosened. A vertical window was added to the spacerto maintain adequate optical access. The spacer sets the internal volumeto 2 mL and enables reproducible production of reactors. A picture ofthe assembled large reactor is shown in FIGS. 12B, 12D, and 12E.

Example 9

This example describes the techniques used to reduce pressure in thereactor. Pressure drop was inherently caused by the resin. Pressure dropwas overcome by employing a rest period after high pressure flows orusing a large reactor.

A low pressure polymer reactor was used, so an overpressure alarm on theHPLC pump was set to shut off the pump at 240 psi, which wasoccasionally triggered. When the alarm was triggered, the system wasallowed to rest for 30 seconds, and the pumps were restarted withoutfurther incident. During this resting phase, the resin visibly expanded.By observing the HPLC pump pressure, it was concluded that if too muchpressure was applied to the beads, they begin to compact. This increasedthe pressure drop across the bed, and the rate of compaction, whichquickly triggers the over pressure alarm. Similar 1% divinyl benzenecrosslinked polystyrene resin available for gel permeationchromatography from Bio-Rad was recommended for gravity drivenseparations only, because it is very soft once swollen.

When the reactor was disassembled immediately after such an event, theresin looked like a solid block and, when probed with a pipette tip,felt like a hard mass. It was difficult to immediately pipette it out.After a few tens of seconds, the resin relaxed and could be pipettedout. A high pressure stainless steel reactor was built and tested, butthe very high pressure necessary to maintain a high flow through acompacted bed (>1000 psi) was reminiscent of previous continuous flowSPPS that struggled with extrusion of the resin through the frit.

It was believed that the initial compaction took place at the boundaryof the frit and the resin, such that the resin was able to mechanicallyblock the pores of a course frit with relatively little deformation. Totest this theory, the original 40 micron frit was replaced with a 20micron frit, and, in more limited trials, 10 micron frits and 2 micronfrits. Smaller pores did not eliminate the problem, but seemed toqualitatively reduce its severity. From this it was concluded that theproblem was inherent in the resin, and can only be eliminated by runningat lower flow rates or reducing the bed height (using smaller scalesand/or larger reactors).

The use of harder, more highly crosslinked resin has been reported, butthe resulting peptides were of inferior quality. The solution used herewas to wait 30 seconds following a high pressure event. This waseffective and expedient, allowing progress on a reasonable scale withoutfurther optimizing the dimensions of the reactor. A limited number oftrials with a ½″ ID reactor (described in Example 8) show nooverpressure with up to 200 mg of resin, operating on the same cycle.

Example 10

This example describes the preparation of ALFALFA-CONHNH₂ in sixminutes. A high capacity pump head for the HPLC pump used in previousexamples was used to deliver 100 ml/min of DMF during the wash step, 100ml/min of 50% piperidine in DMF during the deprotection step, and 12ml/min of activated amino acids during the coupling step. The reactorand preheat loop were maintained at 60° C. by immersion in a water bath.

The apparatus shown in FIG. 13 was constructed. Reservoir 1 containedactivated alanine, reservoir 2 contained activated leucine, reservoirthree contained activated phenylalanine, reservoir 4 contained 50%piperidine in DMF, and reservoir 5 contained DMF. Each activated aminoacid was prepared by combining 50 ml of 0.4M HBTU in DMF with 20 mmolFmoc protected amino acid. Immediately before the start of the run, 10mL of DIEA was added to each of the amino acid reservoirs. To obtain thedesired flow rates, 1.5 bars of nitrogen head pressure was applied toeach reservoir. All tubing upstream of the pump was ⅛″ OD, 1/16″ ID PFA.The three way valves were Swagelok ⅛″ three way valves. The common linesof three way valves were routed into a switching valve (Valco C25-6180)which selected between the reagents. All valves were manuallycontrolled. The pump was a Varian Prostar 210 with a 100 ml/min pumphead. The preheat loop was 1.8 m of 1/16″ OD, 0.030″ ID PFA tubing. Thereactor used was the larger reactor shown in FIG. 12 and described inExample 8. The reactor contained 120 mg of chlorotrityl hydrazidefunctionalized polystyrene resin, prepared from commercial chlorotritylchloride resin using standard methods known to those of ordinary skillin the art. Using the larger reactor helped in maintaining a manageablepressure drop at 100 ml/min.

One synthetic cycle was performed as follows. First a 20 second couplingwas performed at 12 ml/min. The multiport valve was set to the desiredamino acid and the three way valve was set to DMF. All other three wayvalves were set to DMF. After thirty seconds, the selected three wayvalve was switched from amino acid to DMF and the pump flow rate was setto 100 ml/min. After five seconds, the multiport valve was switched topiperidine. After another five seconds, the selected three way valve wasswitched from DMF to piperidine. After 10 seconds the selected three wayvalve was switched back to DMF. After five seconds, the multiport valvewas moved to the next desired amino acid. After another five seconds,the flow rate was reduced to 12 ml/min and the selected three way valvewas switch from DMF to the next desired amino acid, starting the nextcycle. The total time for each step was as follows: 20 second coupling,10 second wash, 10 second deprotection, and 10 second wash. The totaltime for each cycle was 50 seconds. The total ion chromatogram from theLC-MS analysis of the crude material is shown in FIG. 14.

All of the above-listed times are believed to be conservative estimatesof what would be required to achieve 99%+ yields. It is now known thatat a flow rate of 20 ml/min the deprotection is finished in 5 seconds,and it is expected that at 100 ml/min the deprotection requiressubstantially less than 5 seconds. Longer peptides, such as the commonmodel peptide ACP(65-74), could be prepared, for example, by integratingadditional 3-way valves. The general strategy described in this exampleis expected to be viable for the production of any peptide, includingthose produced using the cycles described in Example 1.

Example 11

This example describes an improved synthesis scheme, in which thesynthesis times (relative to Example 1) were substantially reduced. Thecycle time for the synthesis in this example was less than 3 minutes. Toreduce the cycle time relative to Example 1, the wash step was adjusted.All tubing upstream of the pump was replaced with ⅛″ OD 1/16″ ID PFA,and the two valves upstream of the pump were replaced with ⅛″ Swagelokthree way valves. All tubing lengths were minimal. All other systemcomponents were substantially unchanged relative to Example 1. Unlessexplicitly mentioned below, all procedures remained the same, relativeto Example 1.

The larger tubing and a high capacity pump head (maximum 50 ml/min) wereused to deliver DMF and deprotection reagent at 20 ml/min. As expectedbased on FIG. 11, a one minute wash at 20 ml/min proved to be adequatein all cases. Furthermore, a 5 second deprotection step was found to beadequate at these flow rates. The coupling step was unchanged. Thisyielded a total cycle time of 2 minutes 35 seconds to about 2 minutesand 50 seconds, depending on the speed with which the manual steps areperformed. The wash was set at 20 ml/min instead of the maximal 50ml/min because most users have difficulty manually operating the systemat the higher flow rate. It is expected that automation can be used toovercome this human limitation and allow for the implementation ofcycles of about 10 seconds per residue with sufficiently large pumps.

Two chromatograms of peptides made using this cycle are shown in FIG.15. In each case the main peak is the desired product. These are typicalresults for peptides of this length.

Example 12

This example describes in more detail the materials and methods used inExamples 1-11.

2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), 2-(7-Aza-1Hbenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), hydroxybenzotriazole (HOBT), and Na-Fmocprotected amino acids were from Chem-Impex International, IL,NovaBioChem, Darmstadt, Germany, and Peptide Institute, Japan.4-methylbenzhydrylamine functionalized crosslinked polystyrene (MBHAresin) and p-Benzyloxybenzyl alcohol functionalized crosslinkedpolystyrene (Wang resin) were from Anaspec, CA. N,N-Dimethylformamide(DMF), dichloromethane (DCM), diethyl ether, methanol (MeOH) andHPLC-grade acetonitrile were from VWR, PA. Triisopropyl silane (TIPS)and 1,2 Ethanedithiol were from Alfa Aeser, MA. Trifluoroacetic acid(TFA) was purchased from NuGenTec, CA and Halocarbon, NJ. Solvents forLC-MS were purchased from TJ Baker and Fluka. All other reagents werepurchased from Sigma-Aldrich, MO.

Common solvent mixtures used throughout these experiments were: 0.1%(v/v) TFA in water (A), 0.1% (v/v) Formic acid in water (A′), 0.1% (v/v)TFA in acetonitrile (B), and 0.1% (v/v) formic acid in acetonitrile(B′).

All peptides except the ACP(65-74) batch in FIG. 4D were synthesized onthe flow based SPPS system. All peptides except ACP(65-74) batch andACP(65-74) flow RT in FIGS. 4D and 4C were synthesized at 60° C., withreagents preheated immediately before use via a preheat loop (seesynthesizer design). One synthetic cycle consisted of an amino acidexposure step (e.g., amide bond formation, also referred to as couplingin the examples), an amino acid removal step (e.g., removal of thecoupling reagent, also referred to as a wash step in the examples), adeprotection agent exposure step (e.g., Na Fmoc removal, also referredto as deprotection in the examples), and a deprotection agent removalstep (e.g., removal of deprotection reagent and reaction product,piperidine-dibenzofulvene (piperidine-DBF), also referred to as a washin the examples).

Unless noted, coupling was performed by delivering the followingcoupling solution at 12 ml/min (for approximately 30 seconds). Theactivated coupling solution consisted of 2 mmol of Na-Fmoc and sidechain protected amino acid dissolved in 5 ml of 0.4M HBTU in DMF and 1mL of DIEA. Cysteine was dissolved in 5 mL 0.4M HBTU in DMF, 0.687 mLneat DMF, and 0.313 mL DIEA. In both cases, amino acids were dissolvedin HBTU solution up to several hours before use, and DIEA was addedwithin two minutes of use. Volumetric measurements were made at RT (20°C.). The ACP(65-74) shown in FIG. 4A was synthesized by substitutingHATU for HBTU in the above solution.

Next, the coupling solution was removed with 20 mL of DMF delivered at10 mL/min over 2 minutes, and then the Na-Fmoc protection group wasremoved with 3.3 mL of 50% (v/v) piperidine in DMF delivered at 10mL/min over 20 seconds. Excess piperidine and piperidine-DBF wereremoved with 20 mL of DMF delivered at 10 ml/min over 2 minutes tocomplete one cycle.

All peptides were synthesized on 100 mg of 1% divinyl benzenecrosslinked polystyrene resin. To produce C-terminal carboxamidepeptides, MBHA functionalized resin with a loading of 1 mmol per gramwas used, and the first residue coupled was the TFA labile Rink linker.To produce C-terminal hydrazide peptides for ligation, Wang resin,functionalized as below, was used. The loading was 0.6 mmol/g (0.06 mmolscale).

Non-cysteine containing carboxamide peptides were cleaved from the resinand side-chain deprotected by treatment with 2.5% (v/v) water and 2.5%(v/v) TIPS in TFA for two hours. Cysteine containing carboxamidepeptides were cleaved from the resin and side chain deprotected with2.5% (v/v) EDT, 2.5% (v/v) TIPS, and 1% (v/v) water in TFA for twohours. Hydrazide peptides were cleaved with 5% (v/v) EDT, 5% (v/v) TIPS,and 2.5% (v/v) water in TFA for two hours. In all cases, the resin wasremoved and compressed air was used to evaporate the cleavage solutionto dryness at RT. The resulting solids were washed three times with colddiethyl ether, dissolved in 50% A/50% B (v/v), and lyophilized. Sidechain protection was as follows: Arg(Pbf), Tyr(tBu), Lys(Boc),Asp(OtBu), Gln(Trt), Ser(tBu), His(Trt), Asn(Trt), Trp(Boc), Glu(OtBu),Thr(tBu), Cys(Trt).

The Wang resin was functionalized as follows: 5.47 g Wang resin wasadded to a 500 ml round bottom flask and suspended in 98 mL of DCM and1.12 mL of N-Methyl morpholine. This was stirred in an ice bath for 5min and 2.03 g p-nitrophenol chloroformate was added as a powder. Thismixture was stirred for 8.5 hours. The ice in the bath was notreplenished, which allowed the reaction to slowly reach RT. The mixturewas filtered and the solids washed with DCM, DMF, MeOH, and DCM to givea white resin. The resulting resin was placed in a clean 500 ml roundbottom flask in an ice bath, and suspended in a prepared mixture of 210mL DMF, 54 mL DCM, and 1.1 mL hydrazine monohydrate prechilled to 0° C.This yielded a bright yellow solution. The reaction proceeded for 18hours in an ice bath that was allowed to melt. The mixture was thenfiltered, and the solids washed as before to givehydrazine-functionalized Wang resin.

All peptides were analyzed on an Agilent 6520 Accurate Mass Q-TOF LC-MS.For all peptides except GCF and LYRAG, an Agilent C3 Zorbax SB column(2.1 mm×150 mm, 5 μm packing) was used. The flow rate was 0.4 mL/min ofthe following gradient: A′ with 1% B′ for 3 minutes, 1-61% B′ rampinglinearly over 15 min, and 61% B′ for 4 minutes. For GCF and LYRAG, anAgilent C18 Zorbax SB column (2.1 mm×250 mm, 5 μm packing) was used. Theflow rate was 0.4 mL/min of the following gradient: A′ with 1% B′ for 5minutes, 1-61% B′ ramping linearly over 15 min, and 61% B′ for 4minutes.

The peptides were purified as follows. Crude peptides were dissolved in95% A/5% B (v/v) and purified on a Waters preparative HPLC with anAgilent Zorbax SB C18 column (21.2 mm×250 mm, 7 μm packing), a lineargradient from 5%-45% B in A over 80 min, and a flow rate of 10 mL/min.The crude affibody Fragment 1-39 ligation product was purified on aBeckman System Gold semi-preparative HPLC with a Zorbax C18 column (9.4mm×250 mm, 5 μm packing), a linear gradient from 10% to 55% B in A over90 minutes, and a flow rate of 5 mL/min. The final affibody was purifiedon the same system with the same gradient, using a Jupiter C18 column(4.6 mm×250 mm, 5 μm packing) and a flow rate of 2.3 mL/min.

For all purifications, one minute fractions were collected and screenedfor the correct mass on a PerSpective Biosystems Voyager-DE MALDI-TOFusing 2 μL of the fraction co-crystallized with 2 μL of 50% A′/50% B′(v/v) saturated with alpha-cyano-4-hydroxycinnamic acid matrix. Thepurity of pooled fractions was confirmed by LC-MS, as above.

The UV detector response was quantified as follows. To understand the UVtraces produced and the wash efficiencies they represent, the responseof the UV detector was quantified. To determine the approximateconcentration of amino acid in the UV traces, a serial dilution ofFmoc-Ala-OH coupling solution was prepared. The initial concentration ofamino acid was about 0.3M (2 mmol in 6.5 mL total volume) 10×, 100×,1000×, 10,000 and 100,000× dilution standards were prepared and injecteddirectly into the UV detector. The 100× dilution (3×10⁻³M) was justbelow saturation. The 10,000× dilution (3×10⁻⁵M) standard was just abovebaseline, about 1% of scale, as expected. The 100,000× dilution wasbelow the detection limit. The highly reproducible washout traces (FIG.10) show that this is representative of all amino acids (qualitativelydifferent traces between cycles would be expected if the absorbance wasvastly different).

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,and/or methods, if such features, systems, articles, materials, and/ormethods are not mutually inconsistent, is included within the scope ofthe present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

What is claimed is: 1-17. (canceled)
 18. A process for adding amino acidresidues to peptides, comprising: flowing a first stream comprisingamino acids; flowing a second stream comprising an amino acid activatingagent; merging the first and second streams to form a mixed fluidcomprising activated amino acids; and within about 30 seconds aftermerging the first and second streams to form the mixed fluid, exposingthe mixed fluid to a plurality of peptides immobilized on a solidsupport.
 19. The process of claim 18, wherein the amino acid activatingagent comprises an alkaline liquid, a carbodiimide, and/or a uroniumactivating agent. 20-23. (canceled)
 24. The process of claim 18, whereinthe solid support comprises polystyrene and/or polyethylene glycol. 25.The process of claim 18, wherein the solid support comprises a resin.26. The process of claim 25, wherein the solid support comprises amicroporous polystyrene resin.
 27. The process of claim 18, wherein thesolid support is contained within a packed column and/or a fluidizedbed.
 28. The process of claim 18, wherein an amino acid residue is addedto at least about 99% of the immobilized peptides after exposing themixed fluid to the immobilized peptides.
 29. The process of claim 18,wherein multiple copies of the amino acid residue are bonded to fewerthan about 1% of the immobilized peptides after exposing the mixed fluidto the immobilized peptides.
 30. The process of claim 28, wherein addingan amino acid residue to the immobilized peptides comprises adding asingle amino acid residue to the immobilized peptides.
 31. The processof claim 28, wherein adding an amino acid residue to the immobilizedpeptides comprises adding a peptide comprising two or more amino acidresidues to the immobilized peptides. 32-71. (canceled)
 72. The processof claim 18, comprising exposing the mixed fluid to the plurality ofpeptides immobilized on the solid support within about 15 seconds aftermerging the first and second streams to form the mixed fluid.
 73. Theprocess of claim 18, comprising exposing the mixed fluid to theplurality of peptides immobilized on the solid support within about 10seconds after merging the first and second streams to form the mixedfluid.
 74. The process of claim 18, comprising exposing the mixed fluidto the plurality of peptides immobilized on the solid support withinabout 5 seconds after merging the first and second streams to form themixed fluid.
 75. The process of claim 18, wherein the amino acidscomprise protection groups.
 76. The process of claim 75, wherein aminegroups of the amino acids are protected.
 77. The process of claim 75,wherein the protection groups comprise fluorenylmethyloxycarbonylprotection groups.
 78. The process of claim 75, wherein the protectiongroups comprise tertbutyloxycarbonyl protection groups.
 79. The processof claim 75, wherein the protection groups comprise allyloxycarbonylprotection groups.
 80. The process of claim 75, wherein the protectiongroups comprise carboxybenzyl protection groups.